Recombinant Foot and Mouth Disease Vaccine

ABSTRACT

A foot and mouth disease virus (FMDV) vaccine and method for producing same is described wherein the N terminal portion of the FMDV polyprotein, encoding the four structural proteins, 1A, 1B, 1C, and 1D, are each separated by a non-FMDV protease, preferably a cellular protease, for example, furin. The expression system may be transformed into a cell expressing the non-FMDV protease and the resulting particles recovered for use as a vaccine

PRIOR APPLICATION INFORMATION

This application claims the benefit of U.S. Ser. No. 60/635,610, filedDec. 14, 2004 and U.S. Ser. No. 60/643,579, filed Jan. 14, 2005.

BACKGROUND OF THE INVENTION

Foot-and-mouth disease (FMD) is a usually acute disease affectingcloven-hoofed domestic and wild animals like cattle, buffalo, sheep,deer, and pigs. The disease is associated with a high morbidity and lowmortality. Subclinical and persistent infections occur and pose majorproblems for disease control. The virus is highly contagious and istransmitted by contact with infected animals and contaminated materials,humans, and non-susceptible animals. Over the past twenty years, FMD hasbeen endemic in large areas of Asia, Africa, Southern America, andoccasionally Europe, but not in Australia and Northern America. Diseasecontrol measures often included massive culling. This strategy hasreceived intense criticism. Current vaccines for FMD are readilyavailable and safe; however, due to the complex production process andother drawbacks they are not employed as a universal and global weaponagainst FMD.

Outbreaks of FMD result in devastating and drastic consequences for bothanimals and humans. Affected countries suffer from substantial loss inlivestock and animal products, and in export markets, both short- andlong-term. Additional costs, distress and suffering arise fromeradication measurements, compensation policies, and disruption ofnormal living.

Vaccination against FMDV is an established and specific tool to helpcontrol both FMD eradication and outbreaks. Definitive strategies,however, do not exist and depend on a broad range of implications. Thereare no antiviral treatments for FMDV.

FMDV is an antigenically variable virus consisting of seven serotypes(European types A, O and C; African types SAT1, SAT2 and SAT3; and anAsiatic type Asia 1) and dozens of subtypes (see for example Kleid etal., 1981, Science 214: 1125-1129). Immunity to one serotype does notprovide protection against the others and in some cases, immunity to onesubtype will not protect against other members of the same subtype.Currently used vaccines consist of tissue culture grown virus, which forsome preparations are partially purified and which are typicallyinactivated by binary ethyleneimine (BEI).

Modern FMD vaccines, in combination with other measures, can be used tocontain and eradicate FMD outbreaks. Contact transmission of FMDV israpidly reduced within 3-5 days after vaccination of pigs, cattle,sheep, and other animals.

However, some concerns exist over the use of FMD vaccines. Afterexposure to FMDV, vaccination may only prevent disease but notinfection, and some animals may become persistently infected carriers ofFMDV. Using approved diagnostic tests, it is difficult, if notimpossible to differentiate vaccinated from infected or convalescentanimals. Based on the current technology, production of vaccinesrequires handling of live virus in high containment facilities, whichexcludes countries such as the USA from vaccine production.

US Published Patent Application 2004/0001864 teaches a vaccine againstfoot-and-mouth disease wherein empty capsids are produced bycoexpressing P1 and protease 3C.

U.S. Pat. No. 5,864,008 teaches a Th-cell epitope derived from VP3capsid protein of FMDV.

U.S. Pat. No. 5,612,040 teaches a foot-and-mouth disease vaccinecomprising deletion of the G-H loop of VP1 which results in an antigenicbut non-infectious virus.

U.S. Pat. No. 5,824,316 teaches a genetically engineered foot-and-mouthdisease virus wherein the leader proteinase has been deleted. The Lproteinase-deleted viruses are able to assemble and grow in cells inculture, but are less toxic to infected cells within the cells, therebyproducing an attenuated infection.

U.S. Pat. No. 6,048,538 teaches the use of immunodominant domains fromFMDV non-structural proteins 3A, 3B and 3C and the use thereof fordetecting anti-FMDV antibodies in animal body fluids.

Published US Patent Application 2003/0171314 teaches early protection ofsusceptible animals against FMDV by inoculating the animals with avaccine comprising an interferon DNA sequence and optionally afoot-and-mouth disease vaccine.

U.S. Pat. No. 6,107,021 teaches a peptide composition comprising atleast one target antigenic site derived from VP1 capsid protein of FMDVcovalently linked to a helper T cell epitope.

Published PCT Application WO 03/083095 teaches insertion of aheterologous sequence between two furin cleavage sites within a carrierglycoprotein, such as the furin cleavage sites of an F protein of aRespiratory Syncytial Virus and using the resulting mutant virus as amultivalent vaccine.

Published US Patent Application 2004/0001864 teaches the preparation ofempty FMDV capsids by expression of the P1 region and protease 3C. Masonet al. (Vaccines for OIE List A and Emerging Animal Diseases, 2003,Brown and Roth eds, Dev Biol Base1, Karger, vol 114: 79-88) teaches asimilar method, involving the expression of a fragment of the P1 regionand protease 3C for producing empty capsids.

Structures on the surface of the virus particle present antigenic sitesthat are important for the immune response to FMDV. In particular,fragments derived from surface peptide 1D elicit neutralizing antibodiesthat could protect animals from challenge. However, when used alone,1D-based vaccines failed to induce sufficient immunity in challengeexperiments. In general, the entire accessible surface of a virus wouldbe expected to be antigenic and may thus assist in the generation of astrong immunity. Specific formulations of all, recombinantly expressednon-infectious FMDV capsid proteins appear to be as efficacious as acommercial vaccine, with regard to immunogenicity and resistance tochallenge with FMDV.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided anexpression system comprising a promoter operably linked to a nucleicacid molecule encoding a poly-protein, said polyprotein comprising:

FMDV 1A protein-nonFMDV protease recognition sequence-FMDV 1Bprotein-nonFMDV protease recognition sequence-FMDV 1C protein-nonFMDVprotease recognition sequence-FMDV 1D protein.

According to a second aspect of the invention, there is provided amethod of producing a foot and mouth disease virus-like particlecomprising:

providing a host cell including an expression system comprising apromoter operably linked to a nucleic acid molecule encoding apoly-protein, said polyprotein comprising FMDV 1A protein-nonFMDVprotease recognition sequence-FMDV 1B protein-nonFMDV proteaserecognition sequence-FMDV 1C protein-nonFMDV protease recognitionsequence-FMDV 1D protein, said host cell expressing a proteaserecognizing said nonFMDV protease recognition sequence;

growing the host cell under conditions such that the poly-protein isproduced, resolved into 1A, 1B, 1C and 1D by the protease and 1A, 1B, 1Cand 1D assemble into virus-like particles; and

recovering the virus-like particles.

According to a third aspect of the invention, there is provided the useof the virus-like particles prepared as described above as a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the FMDV genome, strain 0, UKGJ3512001, andthe open reading frame for the polyprotein. Protein cleavage sites andthe respective proteases are indicated as arrows.

FIG. 2. Protein chart for the authentic and modified capsidpolyproteins. Amino acid changes of the modified polyprotein areunderlined in blue. These sites represent newly introduced furincleavage sites.

FIG. 3. Hydrophilicity of FMDV capsid proteins.

FIG. 4. Blot showing cleavage of polypeptide by furin. Cells have beentransfected with 3 different plasmids encoding for the original (leftlane; “no cut”) or modified (center and right lane) FMDV capsid (VP1-4).New cleavage sites (A, B, C) have been introduced as indicated (centerand right lane) and utilized. The authentic A cleavage site isrecognized by a cellular protease and partially cleaved (left lane). Themodified FMDV capsids are cleaved to completion at B (center lane) andat A, B, and C (right lane). Since the blot is probed with a monoclonalantibody directed towards VP1 only proteins containing VP1 arevisualised.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference.

As used herein, “cloven-hoofed” animal refers to domestic and wildanimals, for example, but by no means limited to cattle, buffalo, sheep,deer, and pigs.

Non-infectious molecular approaches should preferably offer a safeproduction of vaccines, a platform based technology for all viralstrains, and the inclusion of a marker for distinguishing betweenvaccinated and non-vaccinated animals. That is, one needs to be able todistinguish between vaccinated and infected animals. In one embodiment,as discussed herein, a vaccine or expression system that includes onlyFMDV capsid and not the FMDV polymerase proteins is used so thatvaccinated animals (which will not have anti-polymerase antibodies butwill have anti-capsid antibodies) and infected animals (which will haveboth anti-polymerase antibodies and anti-capsid antibodies) can bedistinguished. In an alternative embodiment, the expression system orvaccine includes a marker gene. As will be appreciated by one of skillin the art, when expressed, the marker gene will produce a detectablemarker, for example, an immune response to the product of the markergene. Other suitable markers known in the art may also be used.

FMDV contains a single-stranded positive sense ribonucleic acid (RNA)genome of approximately 8,300 bases surrounded by an icosahedral capsidcomposed of 60 copies each of four structural proteins, 1A, 1B, 1C, and1D. The RNA genome is translated as a single, long open-reading frameand codes for the four structural proteins and a number ofnon-structural proteins, which function in various aspects of the viruscycle. The viral polyprotein is co-translationally processed by at leastthree viral encoded proteinases (L^(pro), 2A oligopeptide and 3C^(pro)).Most of the cleavages are catalysed by 3CP^(pro) or a3C^(pro)-containing precursor, including the processing of the capsidprecursor polypeptide, P1, into 1AB, 1C, and 1D. One exception is thematuration cleavage of the precursor capsid protein 1AB in the provirionto generate the capsid proteins 1A and 1B that occurs by an unknownmechanism. Only 1D, 1B, and 1C have been shown to be surface-exposed andan immunologically important loop found between the G and H beta strandsof 1D has been identified as a prominent surface structure on the viralcapsid. Cleavage of the polyprotein is essential and sufficient forvirus particle formation.

Described herein is an FMDV vaccine and method for producing samewherein the N-terminal portion of the FMDV polyprotein, encoding thefour structural proteins, 1A, 1B, 1C, and 1D, are expressed from an mRNAhaving at least a 5′ cap and a 3′ poly-A tail, transcribed from a newlymanufactured cDNA. As will be appreciated by one of skill in the art,other modifications to the mRNA and/or cDNA may also be made, forexample, sequence modifications including but by no means limited tothose discussed below and structural modifications known in the art. Theprotein cleavage recognition sites at either ends of the proteins aremodified and are accessible for a non-FMDV protease, preferably acellular protease, for example, Furin. As will be appreciated by one ofskill in the art, the protease cleavage sites are found at the junctionsbetween 1A and 1B, between 1B and 1C and between 1C and 1D. As such, inmost embodiments, the 5′ end of 1A and the 3′ end of 1D will not bemodified.

As used herein, 1A, 1B, 1C and 1D refer to the amino acid sequences (andnucleotide sequences derived therefrom) of these proteins known in theart as discussed herein. Specifically, as shown in Table 1, sequencesfor a number of FMDV isolates are known and as discussed below sequencesfrom these isolates can be used in the invention.

The consensus sequence for furin is Arg-X-Lys/Arg-Arg↓-, (Molloy et al.,1999, Trends in Cell Biology 9: 28-35).

As will be appreciated by one of skill in the art, other suitablecellular proteases may be used. Preferably, the protease cleavage siteis approximately the same length and has a similar hydrophobicity and/orthree dimensional structure and/or charge distribution as the nativeFMDV protease cleavage site.

Thus, in one embodiment of the invention, there is provided anexpression system comprising a nucleic acid molecule encoding apolypeptide comprising 1A, 1B, 1C and 1D of FMDV each separated by anon-native FMDV protease cleavage site. In a preferred embodiment of theinvention, the expression system comprises a suitable promoter operablylinked to a nucleic acid encoding a polypeptide comprising 1A-non-FMDVPCS-1B-non-FMDV-PCS-1C-non-FMDV-PCS-1D, wherein “PCS” refers to“protease cleavage site”. In a yet further embodiment, the nucleic acidencodes a polyprotein comprising 1A-furin PCS-1B-furin PCS-1C-furinPCS-1D.

An example of such a construct is shown in FIG. 2 and in SEQ ID No. 1.As shown in FIG. 4, this construct was cleaved by furin into 1A, 1B, 1Cand 1D proteins and assembled into virus-like particles. As will beapparent to one of skill in the art on examination of the sequence shownin FIG. 2 and SEQ ID No. 1, the FMDV strain is 0 Manisa. A similarconstruct was constructed using 1A, 1B, 1C and 1D proteins from A24Cruzeiro also with furin sites engineered between the proteins andsimilar results were obtained, clearly indicating that the invention canbe used to produce virus-like particles from different FMDV strains, asdiscussed below.

In another embodiment of the invention, there is provided a nucleic acidmolecule encoding a polypeptide having a first domain that has at least80% homology or at least 85% homology or at least 90% homology or atleast 95% homology to amino acids 1-80 of SEQ ID No. 1, a second domainadjacent to the first domain that is a non-FMDV protease cleavage site;a third domain that has at least 80% homology or at least 85% homologyor at least 90% homology or at least 95% homology to amino acids 86-298of SEQ ID No. 1; a fourth domain that is a non-FMDV protease cleavagesite; a fifth domain that has at least 80% homology or at least 85%homology or at least 90% homology or at least 95% homology to aminoacids 304-518 of SEQ ID No. 1; a sixth domain that is a non-FMDVprotease cleavage site; and a seventh domain that has at least 80%homology or at least 85% homology or at least 90% homology or at least95% homology to amino acids 524-733 of SEQ ID No. 1. In a preferredembodiment, the non-FMDV protease cleavage site is a furin cleavage siteas described above.

In another embodiment, there is provided a nucleic acid molecule havingat least 80% homology or at least 85% homology or at least 90% homologyor at least 95% homology to SEQ ID No. 1 and wherein the sequence of thefurin cleavage sites (amino acids 81-81, 299-303 and 519-523 of SEQ IDNo. 1) are such that the cleavage sites are recognized by furin, thatis, are within the furin consensus sequence described above and aretherefore functional for cleavage by furin.

In some embodiments, the expression system further includes a signalsequence for translocating the protein into the ER/golgi apparatus ofthe host cells.

Examples of suitable promoters include but are by no means limited toCMV-driven and baculovirus-driven promoters and vectors. As will beappreciated by one of skill in the art, any suitable promoter active ina cell line expressing the non-FMDV protease of choice may be used,although clearly, the stronger or more efficient the promoter, thehigher the yield of particles.

As will be appreciated by one of skill in the art, any suitable cellline or cell type may be transfected or transformed with the expressionsystem described herein. For example, in those embodiments wherein thenon-FMDV protease is furin, any cell expressing the furin protease maybe used. It is of note that the expression system may be expressed froma transient genetic element such as a plasmid or linear DNA or may beintegrated into the genome of the host cell.

As will be appreciated by one of skill in the art, the cell or cell lineof choice must also express the non-FMDV protease of choice. It is ofnote that the cell or cell line may express the protease naturally or asthe result of genetic manipulation.

Examples of suitable cell lines include but are by no means limited toBHK and 293T.

Specifically, the expression system is transformed or transfected asdiscussed above and the cells are grown under conditions suitable forexpression from the expression system. A poly-protein is produced whichis cleaved by the non-FMDV protease, thereby resolving the poly-proteininto 1A, 1B, 1C and 1D which in turn assemble into a particle. In someembodiments, the particles are secreted from the cell and can berecovered using means known in the art. In other embodiments, theparticles are recovered using other suitable means known in the art.

As discussed herein, the particles can be used as a vaccine forvaccinating animals at risk of developing or contracting foot and mouthdisease. It is of note that as discussed herein, the particles arenon-replicative and can safely be used as a vaccine that accuratelymimics the structure of native FMDV.

Thus, the cellular protease replaces the viral proteases L^(pro), 2Aoligopeptide and 3C^(pro) and an unknown protease. As a result of thisarrangement, non-infectious eukaryotic and prokaryotic expressionsystems can be used to produce empty particles for use as a vaccine.Alternatively, the particles can also be used as diagnostic tools and/orto study old or new substances/drugs/mechanisms for potential antiviralactivity against FMDV.

Referring to FIG. 2 and to SEQ ID No. 1, 1A corresponds to amino acids 1to 80; 1B corresponds to amino acids 86 to 288; 1C corresponds to aminoacids 304 to 518; and 1D corresponds to amino acids 524 to 734. As willbe appreciated by one of skill in the art, these designations arerelative and may vary between different serotypes and sub-types.Furthermore, the specific sequences of 1A, 1B, 1C and 1D are variableamongst serotypes and sub-types, several of which are documented andwell known in the art (see for example, GenBank database accessionnumbers X00429 (A10), X00871(O1K), AJ251476 (A24) and AJ133357 (C SpainOlot) as well as Table 1).

Thus, in one embodiment of the invention, there is provided anexpression system which expresses a fusion comprising 1A, 1B, 1C and 1D,each separated by a protease cleavage site. As will be appreciated byone of skill in the art, the specific amino acid sequence used for 1A,1B, 1C and/or 1D may all be of any of the serotypes or subtypes of FMDV.In other embodiments, a multivalent vaccine may be generated for exampleby using 1A from one serotype or subtype fused to 1B and/or 1C and/or 1Dfrom a different serotype or subtype.

Any suitable adjuvant known in the art may be used in combination withthe vaccine. Examples of suitable adjuvants include but are by no meanslimited to alum, an acrylic or methacrylic acid polymer, or awater-in-oil or oil-in-water emulsion.

In some embodiments, disulphide bridges are added to increase thestability of the empty capsids using means known in the art.

In one embodiment of the invention, a cDNA molecule is generated byRT-PCR amplification of the 5′ end of the FMDV genome, for example,strain 0, although any suitable FMDV could be used, including an unknownserotype or subtype implicated in an outbreak. Specifically, the primersare designed such that the resulting cDNA sequence will include the ORFsfor 1A, 1B, 1C, and 1D. The cDNA is then subcloned into an appropriateexpression vector. In some embodiments, the expression vector may bearranged such that the inserted cDNA includes for example an addition ofan initial methionine, a stop codon and a poly-A tail.

The cDNA is then mutated to introduce cellular protease recognitionsites, for example, furin cleavage sites between 1A-1B, 1B-1C, and1C-1D. The expression vector is then transformed or transfected into asuitable host which is grown under conditions promoting expression ofthe cDNA, which in turn results in the production of empty FMDV capsidparticles which are then recovered and used in the preparation of avaccine.

In an alternative embodiment, the FMDV responsible for the outbreak iscloned or amplified by RT-PCR as discussed above and is sequenced. AcDNA template comprising 1A-PCS-1B-PCS-1C-PCS-1D wherein the 1A, 1B, 1Cand 1D sequences are either derived from consensus sequences or from aspecific FMDV isolate, for example, a common FMDV isolate is thenmutated or otherwise modified so as to substantially correspond orcorrespond verbatim with the 1A, 1B, 1C and/or 1D sequence of the FMDVresponsible for the outbreak. It is of note that a bank of templatecDNAs as described above could be generated and the one with thegreatest sequence homology to the FMDV responsible for the outbreakselected for mutation or modification.

As shown in FIG. 3, FMDV proteins were translated from DNA and analyzedfor hydrophilicity. The capsid polyprotein for FMDV, strain 0, is shownin black, and the modified capsid in red. Red arrows indicate newlyintroduced furin cleavage site. Predictions are calculated using methodsderived from Kyte & Doolittle (KD) and Hopp & Woods (HW), and forsurface exposure (SE). The analysis results are shown in a table formatof numerical values and as a set of line graphs. For each amino acidresidue, numerical values are given for KD, HW, and SE analyses. Allthree methods provide an indication of the hydrophilic character of theenvironment, and the range for each analysis is shown at the top of thecolumn. Three line graphs plot the values calculated for each of thethree analyses. For all three graphs, values above the axis line arehydrophilic or predicted to be exposed at the surface of the protein. KDvalues fall within a range of +4 to −4, with hydrophilic residues havinga negative score. The most hydrophilic reside has a value of −4.5(arginine). On the graphic display, values above the axis line arehydrophilic; values below the axis line are hydrophobic. KD represents acomposite hydrophobicity scale derived from interpretation of freeenergy changes on a water-vapor phase transition and an analysis ofburied side chains. Each value is the average of the values of 5adjacent residues and is plotted at the middle residue. The range ofvalues is approximately ±4 relative units. HW values fall within a rangeof −3 to +3, with hydrophilic residues having a positive score. The mosthydrophilic residues have a value of ±3.0. On the graphic display,values above the axis line are hydrophilic; values below the axis lineare hydrophobic. HW derived hydrophilicity values from a study ofantigenicity and adjusted the values to maximize the accuracy ofpredicting antigenic determinants. Each value is the average of thevalues of 6 adjacent residues and is plotted at the middle point. Therange of values is approximately ±3 relative units. The SE value ispresented as a proportion of the residue, which is exposed on thesurface of the protein. These values fall within a range of 0 to 1.000.The most exposed amino acid has a value of 0.97 (lysine). On the graphicdisplay, peak values, which fall above the axis line, are predicted tobe exposed on the surface of the protein. SE analysis uses the data ofJanin, et al., which provides values representing the fraction ofresidues of a given amino acid that have a surface area of greater than20 angstroms squared. High values therefore represent amino acids thatare likely to be exposed on the surface of the protein. Plotted valuesare the average of 6 residues and are plotted at the middle point. Basedon these predictions, the three-dimensional structure, the stability,and the immunogenicity of the modified capsid protein are expected to bevery similar to the authentic FMDV, strain 0, capsid protein.

As will be appreciated by one of skill in the art, the native FMDVprotease is of viral origin and would need to be co-expressed with theexpression system expressing the capsid. This approach is inefficientdue to the requirement for co-expression of both capsid proteins andprotease in a single cell and would result in lower yield.

While the preferred embodiments of the invention have been describedabove, it will be recognized and understood that various modificationsmay be made therein, and the appended claims are intended to cover allsuch modifications which may fall within the spirit and scope of theinvention.

Table 1 Exemplary FMDV Sequences

Foot-and-mouth disease virus A isolate a12valle 119 iso20, completegenome gi|46810760|gb| AY593752.1|[46810760]Foot-and-mouth disease virus A isolate a13brazil iso75, complete genomegi|46810762|gb| AY593753.1|[46810762]Foot-and-mouth disease virus A isolate a14 spain iso39, complete genomegi|46810764|gb| AY593754.1|[46810764]Foot-and-mouth disease virus A isolate a15thailand iso43, completegenome gi|46810766|gb| AY593755.1|[46810766]Foot-and-mouth disease virus A isolate a16belem iso80, complete genomegi|46810768|gb| AY593756.1|[46810768]Foot-and-mouth disease virus A isolate a17 Aguarulbos iso83, completegenome gi|46810770|gb| AY593757.1|[46810770]Foot-and-mouth disease virus A isolate a18 zulia iso40, complete genomegi|46810772|gb| AY593758.1|[46810772]Foot-and-mouth disease virus A isolate a1b AYern iso41, complete genomegi|46810774|gb| AY593759.1|[46810774]Foot-and-mouth disease virus A isolate a20ussr iso10, complete genomegi|46810776|gb| AY593760.1|[46810776]Foot-and-mouth disease virus A isolate a21kenya iso77, complete genomegi|46810778|gb| AY593761.1|[46810778]Foot-and-mouth disease virus A isolate a22iraq-95 iso95, complete genomegi|46810780|gb| AY593762.1|[46810780]Foot-and-mouth disease virus A isolate a22iraq64 iso86, complete genomegi|46810782|gb| AY593763.1|[46810782]Foot-and-mouth disease virus A isolate a22iraq70 iso92, complete genomegi|46810784|gb| AY593764.1|[46810784]Foot-and-mouth disease virus A isolate a22turkey iso66, complete genomegi|46810786|gb| AY593765.1|[46810786]Foot-and-mouth disease virus A isolate a23kenya iso8, complete genomegi|46810788|gb| AY593766.1|[46810788]Foot-and-mouth disease virus A isolate a24 argentina iso9, completegenome gi|46810790|gb| AY593767.1|[46810790]Foot-and-mouth disease virus A isolate a24cruzeiro iso71, completegenome gi|46810792|gb| AY593768.1|[46810792]Foot-and-mouth disease virus A isolate a25 argentina iso38, completegenome gi|46810794|gb| AY593769.1|[46810794]Foot-and-mouth disease virus A isolate a26arg iso74, complete genomegi|46810796|gb| AY593770.1|[46810796]Foot-and-mouth disease virus A isolate a27columbia iso78, completegenome gi|46810798|gb| AY593771.1|[46810798]Foot-and-mouth disease virus O strain Akesu/58, complete genomegi|21239433|gb| AF511039.1|[21239433]Foot-and-mouth disease virus O strain China/1/99(Tibet), complete genomegi|21542501|gb|AF506822.21[21542501]Foot-and-mouth disease virus O, strain TAW/2/99 TC, complete genomegi|30145772|emb|AJ539136.1|FOO539136[30145772]Foot-and-mouth disease virus O, strain TAW/2/99 BOV, complete genomegi|30145774|emb|AJ539137.1|FOO539137[30145774]Foot-and-mouth disease virus O, strain Tibet/CHA/99, complete genomegi|30145776|emb|AJ539138.1|FOO539138[30145776]Foot-and-mouth disease virus O, strain SKR/2000, complete genomegi|30145778|emb|AJ539139.1|FOO539139[30145778]Foot-and-mouth disease virus O, strain SAR/19/2000, complete genomegi|30145780|emb|AJ539140.1|FOO539140[30145780]Foot-and-mouth disease virus O, strain UKG/35/2001, complete genomegi|30145782|emb|AJ539141.1|FOO539141[30145782]Foot-and-mouth disease virus HKN/2002, complete genome gi|33348772|gb|AY317098.1|[33348772]Foot-and-mouth disease virus O strain OMIII, complete genomegi|33943915|gb|AY359854.1|[33943915]Foot-and-mouth disease virus O isolate O/NY00, complete genomegi|37575129|gb| AY333431.1|[37575129]Foot-and-mouth disease virus polyprotein gene, genomic RNA, serotype O,isolate FRA/1/2001, complete genomegi|45725010|emb|AJ633821.1|[45725010]Foot-and-mouth disease virus O isolate o10phil54 iso54, complete genomegi|46810878|gb| AY593811.1|[46810878]Foot-and-mouth disease virus O isolate o10phil76 iso76, complete genomegi|46810880|gb| AY593812.1|[46810880]Foot-and-mouth disease virus O isolate o11Indonesia iso52, completegenome gi|46810882|gb| AY593813.1|[46810882]Foot-and-mouth disease virus O isolate o1argentina iso5, complete genomegi|46810884|gb| AY593814.1|[46810884]Foot-and-mouth disease virus O isolate o1bfs iso18, complete genomegi|46810886|gb| AY593815.1|[46810886]Foot-and-mouth disease virus O isolate o1bfs46 iso46, complete genomegi|46810888|gb| AY593816.1|[46810888]Foot-and-mouth disease virus O isolate o1brugge iso79, complete genomegi|46810890|gb| AY593817.1|[46810890]Foot-and-mouth disease virus O isolate o1campos iso96, complete genomegi|46810892|gb| AY593818.1|[46810892]Foot-and-mouth disease virus (FMDV) strain C, isolate c-s8c1, genomicRNA gi|6318187|emb|AJ133357.1|FDI133357[6318187]Foot-and-mouth disease virus (FMDV) strain C, isolate rp99, genomic RNAgi|6318189|emb|AJ133358.11FAN133358[6318189]Foot-and-mouth disease virus (FMDV) strain C, isolate rp146, genomic RNAgi|6318191|emb|AJ133359.11FAN133359[6318191]Foot-and-mouth disease virus C1 isolate c1noville iso56, complete genomegi|46810864|gb| AY593804.1|[46810864]Foot-and-mouth disease virus C1 isolate c1ober iso88, complete genomegi|46810866|gb| AY593805.1|[46810866]Foot-and-mouth disease virus C3 isolate c3ind iso19, complete genomegi|46810868|gb| AY593806.1|[46810868]Foot-and-mouth disease virus C3 isolate c3resende iso1, complete genomegi|46810870|gb| AY593807.1|[46810870]Foot-and-mouth disease virus C4 isolate C4 Tierra del Fuego iso2,complete genome gi|46810872|gb| AY593808.1|[46810872]Foot-and-mouth disease virus C5 isolate c5arg iso60, complete genomegi|46810874|gb| AY593809.1|[46810874|Foot-and-mouth disease virus C isolate cwald iso32, complete genomegi|46810876|gb| AY593810.1|[46810876]Foot-and-mouth disease virus Asia1 strain YNBS/58, complete genomegi|37223495|gb| AY390432.1|[37223495]Foot-and-mouth disease virus Asia 1 isolate asia1-1pak iso3, completegenome gi|46810846|gb| AY593795.1|[46810846]Foot-and-mouth disease virus Asia 1 isolate asia1-2isrl3-63 iso6,complete genome gi|46810848|gb| AY593796.1|[46810848]Foot-and-mouth disease virus Asia 1 isolate asia1-3kimron iso61,complete genome gi|46810850|gb| AY593797.1|[46810850]Foot-and-mouth disease virus Asia 1 isolate asia1leb-89 iso89, completegenome gi|46810852|gb| AY593798.1|[46810852]Foot-and-mouth disease virus Asia 1 isolate asia1leb4 iso4, completegenome gi|46810854|gb| AY593799.1|[46810854]Foot-and-mouth disease virus Asia 1 isolate asia1leb83 iso28, completegenome gi|46810856|gb| AY593800.1|[46810856]Foot-and-mouth disease virus Asia 1 isolate IND 321/01, complete genomegi|51340579|gb| AY687333.1|[51340579]Foot-and-mouth disease virus Asia 1 strain IND 491/97, complete genomegi|51340581|gb| AY687334.1|[51340581]Foot-and-mouth disease virus SAT 1 isolate sat1-20 iso11, completegenome gi|46810934|gb| AY593839.1|[46810934]Foot-and-mouth disease virus SAT 1 isolate sat1-3swa iso14, completegenome gi|46810936|gb| AY593840.1|[46810936]Foot-and-mouth disease virus SAT 1 isolate sat1-4srhod iso24, completegenome gi|46810938|gb| AY593841.1|[46810938]Foot-and-mouth disease virus SAT 1 isolate sat1-5sa iso13, completegenome gi|46810940|gb| AY593842.1|[46810940]Foot-and-mouth disease virus SAT 1 isolate sat1-6swa iso16, completegenome gi|46810942|gb| AY593843.1|[46810942]Foot-and-mouth disease virus SAT 1 isolate sat1-7isrl iso12, completegenome gi|46810944|gb| AY593844.1|[46810944]Foot-and-mouth disease virus SAT 1 isolate sat1bot iso47, completegenome gi|46810946|gb|AY593845.1|[46810946]Foot-and-mouth disease virus SAT 1 isolate sat1rhod iso33, completegenome gi|46810948|gb| AY593846.1|[46810948]Foot-and-mouth disease virus SAT2 genomic RNA for L, VP4, VP2, VP3, VP1,2A, 2B, 2C, 3A, VPg1, VPg2, VPg3, pro coding polpolyprotein, strainKEN/3/57 gi|6572136|emb|AJ251473.1|FD1251473[6572136]Foot-and-mouth disease virus SAT 2 clone ZIM/7/83, complete genomegi|33332022|gb| AF540910.1|[33332022]Foot-and-mouth disease virus SAT 2 isolate sat2-1 rhod iso26, completegenome gi|46810950|gb| AY593847.1|[46810950]Foot-and-mouth disease virus SAT 2 isolate sat2-2 iso25, complete genomegi|46810952|gb| AY593848.1|[46810952]

1. An expression system comprising a promoter operably linked to anucleic acid molecule encoding a poly-protein, said polyproteincomprising: FMDV 1A protein-nonFMDV protease recognition sequence-FMDV1B protein-nonFMDV protease recognition sequence-FMDV 1C protein-nonFMDVprotease recognition sequence-FMDV 1D protein.
 2. The expression systemaccording to claim 1 wherein the nonFMDV protease recognition sequenceis a furin protease recognition sequence.
 3. A method of producing afoot and mouth disease virus-like particle comprising: providing a hostcell including an expression system comprising a promoter operablylinked to a nucleic acid molecule encoding a poly-protein, saidpolyprotein comprising FMDV 1A protein-nonFMDV protease recognitionsequence-FMDV 1B protein-nonFMDV protease recognition sequence-FMDV 1Cprotein-nonFMDV protease recognition sequence-FMDV 1D protein, said hostcell expressing a protease recognizing said nonFMDV protease recognitionsequence; growing the host cell under conditions such that thepoly-protein is produced, resolved into 1A, 1B, 1C and 1D by theprotease and 1A, 1B, 1C and 1D assemble into virus-like particles; andrecovering the virus-like particles.
 4. The method according to claim 3wherein the protease is furin.
 5. Use of the virus-like particlesprepared according to claim 3 as a vaccine.